Abstract. Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H] -mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78 % of the 3H-marmose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.T HE processing of asparagine-linked oligosaccharides from high mannose-type units to mature complextype structures occurs in a highly ordered series of reactions (21,27). Enzymes involved in the processing reactions reside within the ER and Golgi apparatus, and biochemical as well as morphological studies indicate that they are restricted to defined compartments along the secretory pathway (13,14,16,18,32,46,47). The physical location of an enzyme within the pathway generally corresponds with the temporal order in which it functions (21, 27). In a previous study (16) we took advantage of the ordered processing reactions to define the intracellular compartments that conmined mutant forms of the G protein of vesicular stomatitis virus (VSV). The mutant G proteins were synthesized in COS-1 cells after transfection with plasmids that coded for proteins whose carboxy termini possessed deletions or alterations. As a result of the alterations, the proteins accumulated at intracellular sites rather than at the cell surface (43,44). The structure of the oligosaccharides attached to the mutant G proteins reflected the site of accumulation within the cells. For example, the G protein encoded by A1473 was shown to accumulate in the ER by immunofluorescence (44), and its asparagine-linked oligosaccharides were processed to high mannose-type units containing eight mannose residues (16...

Monoclonal antibody (mAb) 5B4 recognizes in the rat a large, developmentally regulated membrane glycoprotein. The larger form of this antigen (185-255 kD) occurs in the developing nervous system and is present in membranes of nerve growth cones, as determined by analysis of a growth cone particle fraction. An immunochemical characterization of this antigen and of a smaller form (140 kD), sparsely present in the mature nervous system, has been described (Ellis, L., I. Wallis, E. Abreu, and K. H. Pfenninger, 1985, J. Cell. Biol., 101:1977-1989. The present paper reports on the localization by immunofluorescence of 5B4 antigen in cultured cortical neurons, developing spinal cord, and the mature olfactory system. In culture, mAb 5B4 stains only neurons; it is sparsely present in neurons at the onset of sprouting while, during sprouting, it appears to be concentrated at the growth cone and in regions of the perikaryon. In the developing spinal cord, 5B4 labeling is faintly detectable on embryonic day 11 but is intense on fetal day 13. At this stage, the fluorescence is observed in regions of the cord where axonal growth is occurring, while areas composed of dividing or migrating neural cells are nonfluorescent. With maturation of the spinal cord, this basic pattern of fluorescence persists initially, but the staining intensity decreases dramatically. In the adult, faint fluorescence is detectable only in gray matter, presumably indicating the presence of the 140 kD rather than the fetal antigen. The only known structure of the adult mammalian nervous system where axonal growth normally occurs is the olfactory nerve, mAb 5B4 intensely stains a variable proportion of olfactory axons in the mucosa as well as in the olfactory bulb. Based on both immunochemical and immunofluorescence data, the 5B4 antigen of 185-255 kD is associated specifically with growing neurons, i.e., neurons that are generating neurites.In the previous article (3), the generation of a monoclonal antibody (mAb) j to a neuronal membrane antigen that is developmentally regulated and concentrated in growth cone membranes was reported. In the brain of the fetal rat, this antibody (mAb 5B4) recognizes a 185-255-kD membrane glycoprotein that is rich in sialic acid residues and resembles in its biochemical properties the previously described neuronal antigen(s) designated D2 (8, 10), N-CAM (for neural cell adhesion molecule) (e.g., 2, 7, 12), or BSP-2 (4, 6, 13). In the mature brain, mAb 5B4 does not seem to bind to synaptosomal membranes but recognizes a very small amount of a ~ Abbreviations used in this paper. mAb, monoclonal antibody; N-CAM, neural cell adhesion molecule. 1990140-kD protein in crude membrane preparations.In the present paper, we present results obtained by immunofluorescence in order to address the following questions. (a) Is the localization of the 5B4 antigen in the developing brain consistent with the data obtained by subeellular fractionation? (b) How is the expression of the 5B4 antigen related to neurogenesis? (c) How is ...

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